| The prevalence of diabetes mellitus (DM) has increased rapidly in recent years. Diabetic macrovascular complications (DMI), such as coronary heart disease and stroke, are the most devastating clinical manifestations of diabetes. The leading cause of diabetic macrovascular complications are atherosclerotic plaques rupture whieh are characterized by injury of endothelium and exposure of thrombogenic lipid core into the bloodstream. The current treatments are limited in their overall effectiveness. The lipid-lowering and anti-platelet drug treatments are not sufficient to stabilize vulnerable plaques, and intervention therapies might result in re-narrowing. Thus, it is crucial to find new approaches to reduce atherosclerotic plaques rupture, and eventually reduce the disease burden.Endothelial cells (ECs) play a crucial role in the formation and stabilization atherosclerotic plaques. High glucose induces endothelial cell dysfunction and damages the integrity of the endothelium. Circulating LDL cholesterol crosses the damaged endothelium and accumulates in the wall of the artery, which initiate the plaque formation. During the development of atherosclerotic plaques, apoptosis of ECs over the plaques leads to enlargement of the lipid core, loss of collagen and intimal inflammation. Endothelial progenitor cells (EPCs) are a type of bone marrow (BM)-derived precursor cells that can differentiate ex vivo to an endothelial phenotype. Upon endothelial cell dysfunction, EPCs from BM move into the circulation and replace the damaged cells. However, mobilization of EPCs from BM to the atherosclerotic plaques is very limited at non-treatment condition. Thus, interventions improving EPCs recruitment may present a novel strategy for plaques stabilization.Chemokine receptor 5 (CCR5) is a member of the β-chemokine receptor family, and a G-coupled seven-transmembrane chemokine receptor. CCR5 is expressed in leukocytes and monocytes/macrophage. Genetic inactivation of CCR5 is associated with the reduction of pro-atherogenic cytokines and the accumulation of monocytes/macrophages in atherosclerotic plaques. CCR5’s cognate ligand chemokine ligand 5 (CCL5), also known as regulated on activation normal T expressed and secreted (RANTES), is a member of the CC-chemokine family stored in and released from platelets and activated T cells. CCL5 is up-regulated in the injured vessels via activation by platelets during the process of diabetic macrovascular complications. Increased expression of CCL5 on the surface-adherent platelets mediates trafficking of monocytes/macrophages into injured vessels by binding with its receptor CCR5. To date, the effects of CCR5 on stability of atherosclerotic plaques have not yet been addressed. Recent studies reported that CCR5 mediates glomerular microvascular endothelial regeneration by stimulating the adhesion of BM-derived EPCs, and inhibition of CCR5 expression reduces EPCs recruitment during wound healing in mice. Thus, we hypothesize that increased expression of CCR5 in EPCs may enhance the stability of plaques by stimulation of EPCs mobilization and recruitment.Part I Chemokine CCR5 facilitates endothelial progenitor cells recruitment to vascular injury sitesOBJECTIVE To investigate the role of CCR5 on EPCs recruitment to endothelial injury sitesMETHODS1. Animal modelThe type 2 diabetes mellitus models of splenectomized ApoE-/- CSVBL/6J mice were used to perform the experiments. EPCs were isolated from 8 weeks old male C57 mice.2. Splenectomy surgeryApoE-/- mice (8-10 weeks old) were anesthetized with i.p. administration of 0.8% pentobarbital sodium (10 mg/kg body weight). An incision of 10-15 mm was made above the left abdomen of each mouse, and the spleen was removed after cauterizing the splenic arteries and venous supply. The mice were then allowed to recover for 7 days before further treatment.3. Type 2 diabetes mellitus modelSplenectomized ApoE-/- mice with i.p. administration of streptozocin (35 mg/kg body weight), continuous treatment for three days. ApoE-/- mice with i.p. administration of sodium citrate as control groups. In the current study, ApoE-/- mice with a blood glucose level> 300 mg/dl were considered type 2 diabetes mellitus.4. EPCs isolation, culture, and characterizationEPCs were isolated from 8 weeks old male C57 mice. In brief, whole bone marrow cells were collected under sterile conditions from femur (and tibias) by flushing the shaft with PBS. The mononuclear cells were isolated by percoll density gradient centrifugation at 2000g for 20 minutes. After rinsing three times, the isolated cells were cultured in EBM-2 medium with MV Bullet Kit. After 7 days of culture, the EPCs markers Dil-AcLDL and BS-1 lectin were confirmed by immunofluorescence analysis.5. Lentivirus constructs and transfectionThe recombinant lentiviruses (Lenti) carrying murine CCR5 (Lenti-EGFP-CCR5) or a control transgenic EGFP (Lenti-EGFP) were prepared as previously described. The passage 2 of EPCs was used for lentivirus transfection. Prior to transfection, cultured EPCs were labeled with CM-Dil (4 mg/ml, Molecular Probes, Eugene, OR, USA) for 15 minutes according to the manufacturer’s protocol. Then the EPCs were incubated in EBM-2 media containing Lenti-EGFP-CCR5 or Lenti-EGFP particles. The lentiviral particles were removed after 24 hours. The double-labeled EPCs were harvested (passage 3) and resuspended at 1× 105 cells/ml in PBS for administration to the mice.6. Experimental designFourty splenectomized type 2 diabetes ApoE-/- C57BL/6J male mice were treated with a high fat, high glucose, high-cholesterol diet (21% anhydrous milk fat/butter fat,34% sucrose and 0.2% cholesterol; Harlan, Teklad) for 24 weeks. Then the animals were randomly divided into 3 groups and intravenously injected with 200 μl sterile PBS (control group) or 200 μl 1× 106EPCs transfected with Lenti-EGFP (Lenti-EGFP group) or 200 μl 1× 106EPCs infected with Lenti-EGFP-CCR5 (Lenti-CCR5 group). After treatment, all mice were returned to normal chow diet for the remaining experiments.7. Immunofluorescence analysisThe accumulation of EPCs over the atherosclerotic plaques was evaluated by immunofluorescence analysis. In brief, double labeled EPCs (EGFP and CM-Dil) were transplanted into the ApoE-/- mice, and the mice were sacrificed on week 1 and 6 after transplantation. The aortic roots was excised, and 6μm frozen sections were prepared as described previously. The frozen sections were then stained with DAPI for 5 minutes. The number of triple-color positive cells (EGFP+, Dil+, DAPI+) on atherosclerotic plaques were counted in 3 randomly chosen high-power fields (magnification, x200), and representative results from 3 independent experiments are shown.8. Migration and proliferation assaysThe migration of EPCs was evaluated using transwell chambers. The proliferation of EPCs was examined by the 5-ethynyl-2’-deoxeuridine (EdU) assay kit.9. Statistical analysisStatistical differences between groups were examined by one-way ANOVA with Mann-Whitney’s U test. A P< 0.05 was considered as statistically significant. All statistical analyses were performed using SPSS 18.0 software.RESULTS:1. Detection of the restructuring lentivirus interference vectorsThe restructuring CCR5 lentivirus expressing vector was examined using sequencing validation. The virus tilter of EGFP is 3?0010 ifu/ml, and the verus tilter of CCR5 overexpressing vector is 2.8？0010 ifu/ml.2. Identification of BM derived of EPCsAfter 7 days of culture, the EPCs markers DiI-AcLDL and BS-1 lectin were identified by immunofluorescence analysis, and more than 90% cells uptaked Dil-acLDL and BS-1 lectin, indicating EPCs was successfully isolated and cultured.3. The transfection efficiency of EPCsThe efficiency of the transfection in EPCs were evaluated by immunofloresence and more than 80% EPCs transfected with lentivirus vectors are positive for EGFP, indicating that EPCs with EGFP or EGFP-CCR5 expression were successfully established. Western blot analysis displayed that the protein level of CCR5 was remarkably higher in the cells of the Lenti-CCR5 group than those of Lenti-EGFP group.4. Overexpression of CCR5 increased EPC recruitment to vascular injury sitesThe accumulation of EPCs over the atherosclerotic plaques in type 2 diabetes mellitus mice was analyzed by the triple-color immunofluorescence (EGFP+, Dil+, and DAPI+). The number of triple-color labeled EPCs detected in the atherosclerotic plaques of the Lenti-CCR5 group is significantly higher than that of the Lenti-EGFP group 7 days (P<0.05) and 6 weeks post-treatment (P<0.01), suggesting that CCR5 overexpression enhanced recruitment of EPCs to endothelial injury sites.5. CCL5/CCR5 interaction involves in EPC migrationWe performed in vitro chemotaxis assays with CCR5 overexpressing EPCs. CCL5 significantly enhanced the migration and adhesion of EPCs in Lenti-CCR5 group than Lenti-EGFP group (P<0.01, P<0.05). After addition of anti-CCL5 antibody, the number of migrated EPCs was similar in the Lenti-EGFP group and Lenti-CCR5 group (.P=0.04), suggesting that CCR5 overexpression induced EPC migration is in a CCL5-dependent manner. Furthermore, we examined EPCs proliferation by EdU incorporation, and the cell growth was similar between the Lenti-CCR5 group and the Lenti-EGFP group (P=0.98).CONCLUSIONS:1. Overexpression of CCR5 increased EPCs recruitment to atherosclerotic plaques.2. Overexpression of CCR5 promotes migration of EPCs without affecting their proliferation.Part II Overexpressing CCR5 on endothelial progenitor cells promotes the stabilization of atherosclerotic plaques in ApoE-/- miceOBJECTIVE:To investigate the therapeutic potential of CCR5 overexpressing EPCs on plaques stabilization in ApoE-/- mouse model.METHODS1. Animal modelSplenectomized ApoE-/- C57BL/6J mice were used to perform the experiments. EPCs were isolated from 8 weeks old male C57 mice.2. Splenectomy surgeryApoE-/- mice (8-10 weeks old) were anesthetized with i.p. administration of 0.8% pentobarbital sodium (10 mg/kg body weight). An incision of 10-15 mm was made above the left abdomen of each mouse, and the spleen was removed after cauterizing the splenic arteries and venous supply. The mice were then allowed to recover for 7 days before further treatment.3. EPCs isolation, culture, and characterizationEPCs were isolated from 8 weeks old male C57 mice. In brief, whole bone marrow cells were collected under sterile conditions from femur (and tibias) by flushing the shaft with PBS. The mononuclear cells were isolated by percoll density gradient centrifugatipn at 2000g for 20 minutes. After rinsing three times, the isolated cells were cultured in EBM-2 medium with MV Bullet Kit. After 7 days of culture, the EPCs markers Dil-AcLDL and BS-1 lectin were confirmed by immunofluorescence analysis.4. Lentivirus constructs and transfectionThe recombinant lentiviruses (Lenti) carrying murine CCR5 (Lenti-EGFP-CCR5) or a control transgenic EGFP (Lenti-EGFP) were prepared as previously described. The passage 2 of EPCs was used for lentivirus transfection. Prior to transfection, cultured EPCs were labeled with CM-Dil (4 mg/ml, Molecular Probes, Eugene, OR, USA) for 15 minutes according to the manufacturer’s protocol. Then the EPCs were incubated in EBM-2 media containing Lenti-EGFP-CCR5 or Lenti-EGFP particles. The lentiviral particles were removed after 24 hours. The double-labeled EPCs were harvested (passage 3) and resuspended at 1 × 106 cells/ml in PBS for administration to the mice.5. Experimental designSixty splenectomized ApoE-/- C57BL/6J male mice were treated with a high fat, high glucose, high-cholesterol diet (21% anhydrous milk fat/butter fat,34% sucrose and 0.2% cholesterol; Harlan, Teklad) for 24 weeks. Then the animals were randomly divided into 3 groups and intravenously injected with 200 μl sterile PBS (control group) or 200 μl 1×106 EPCs transfected with Lenti-EGFP (Lenti-EGFP group) or 200 μ1 1× 106EPCs infected with Lenti-EGFP-CCR5 (Lenti-CCR5 group). After treatment, all mice were returned to normal chow diet for the remaining experiments.6. ImmunohistochemistryThe aortic sinus frozen sections were stained with Oil-red O and the positive staining area was used to quantitate atherosclerotic plaque area. The content of smooth muscle cells, monocytes/macrophages, endothelial cells, IL-6 and MMP9 in atherosclerotic plaques were assayed by specific immunostaining.7. ImmunochemistryVenous blood samples of the mice were collected 0 and 6 weeks after treatment, and centrifuged to collect the serum. Inflammatory cytokines were assessed by a mouse atherosclerosis antibody array I, which consists of 22 antibodies for atherosclerosis related cytokines. The Cluster version 3.0 and Java Tree View version 1.60 software were used to analyze the results as described previously. Fold-change (FC) was used to identify genes with large shifts between the control group and treatmeni group. Serum nitrite and nitrate were measured by a colorimetric NO metabolite detection kit. Serum total cholesterol, low density lipoprotein (LDL), triglycerides (TG) and high-density lipoprotein (HDL) were measured by colorimetric enzymatic procedures.8. Statistical analysisStatistical differences between groups were examined by one-way ANOVA with Mann-Whitney’s U test. A P< 0.05 was considered as statistically significant. All statistical analyses were performed using SPSS 18.0 software.RESULTS1. The expression of CCL5 in ascending atherosclerotic aorta of AS patients was examined by immunohistochemistry. The positive staining of CCL5 was observed in the arteries of the patients with no visible atherosclerotic plaques (intima,9.93 ± 4.57%; media,0.43 ± 0.29%; adventitia:17.91 ± 2.66%), arteries of the patients with atherosclerotic plaques (intima,22.18 ± 3.37%; media,23.87±5.04 %, adventitia:28.31 ± 4.42%), and arteries of the patients with advanced unstable plaques (intima,23.38 ± 3.23%; media,22.42 ± 9.02%; adventitia:43.37±0.67 %). Quantitative image analysis indicated a significant increase of CCL5 expression in the intima and media of the patients with advanced unstable plaques than those in no visible atherosclerotic plaques (intima, P<0.05; media, P<0.01). However, no significant difference was found between the arteries of the patients with advanced unstable plaques and the arteries of the patients with atherosclerotic plaques (intima, P=0.64; media, P=0.81). CCL5 expression in the adventitia was significantly increased in the arteries of the patients with advanced unstable plaques than those in atherosclerotic plaques or no visible atherosclerotic plaques (P<0.05, P<0.01, respectively). CCL5 expression was also examined in ApoE-/-mice fed with high fat diet. The positive staining area of CCL5 was observed in the arteries of the mice with no visible atherosclerotic plaques (intima,20.23±2.35 %; media,19.86± 4.75%; adventitia:3.15±0.30%), arteries of the mice with atherosclerotic plaques (intima,33.09± 3.91%; media,14.41 ± 4.21%; adventitia: 3.33±0.30%), and arteries of the mice with advanced unstable plaques (intima, 17.43 ± 2.22%; media,36.73 ±0.45%; adventitia:13.5±1.41%). We found a significant decrease of CCL5 expression in the intima of the mice with advanced unstable plaques than those in atherosclerotic plaques (P<0.01). However, there were no significant differences between the arteries of the mice with advanced unstable plaques and the arteries of the mice with no visible atherosclerotic plaques (P=0.20). CCL5 expression was significantly increased in the media of the mice with advanced unstable plaques than those in atherosclerotic plaques or no visible atherosclerotic plaques (P<0.01, P<0.05, respectively). In the adventitia, the expression of CCL5 was significantly increased in the arteries of the mice with advanced unstable plaques than those in atherosclerotic plaques or no visible atherosclerotic plaques (P<0.01, P<0.01, respectively). In addition, we examined the expression of CCR5 in ascending aorta of human and mice. CCR5 protein was faintly detected in non-diseased artery, and was highly expressed in the atherosclerotic plaques in human and mice.2. To examine incorporation of labeled putative EPCs into endothelium, the cross-sections of aortic roots of ApoE-/-mice treated with EPCs were stained with the antibody for CD31, the endothelial cell specific marker. EPCs (EGFP+) were localized in the endothelium of the atherosclerotic plagues, suggesting that EPCs successful engrafted in the vascular wall. EPCs (EGFP+) were also found in sub-endothelial region of the atherosclerotic plagues in Lenti-CCR5 group. In addition, the number of triple-color labeled EPCs detected in the atherosclerotic plaques of the Lenti-CCR5 group is significantly higher than that of the Lenti-EGFP group 1 week (13.5±1.1 vs.6.5±0.7, P<0.05) and 6 weeks post-treatment (13.0±1.4 vs. 2.3±0.5, P<0.01), suggesting that CCR5 overexpression enhanced recruitment of EPCs over the atherosclerotic plaques.3. Six weeks after the treatment, the plaques in the Lenti-CCR5 group (9.58 ± 1.51%) displayed a substantial regression in overall plaques area compared to the Lenti-EGFP (18.72 ± 2.52%) or control group (19.75 ± 2.98%) as quantified by Oil-red O staining of the aortic sinus. Quantitative image analysis showed a significant decrease in the positive staining area of MOMA-2 in Lenti-CCR5 group (2.56± 0.94%) compared with Lenti-EGFP group (7.76±2.37%, P<0.05) or control group (11.4± 1.78%, P<0.01). However, no significant difference was found for the plaques contents of SMC between the Lenti-CCR5 (P=0.76) or Lenti-EGFP (P=0.97) and the control group. Taken together, treatment with CCR5 overexpressing EPCs reduces lipid deposition and macrophages invasion in ApoE" ’’mice.4. The administration of CCR5 overexpressing EPCs (Lenti-CCR5group) significantly decreased the plasma cholesterol and LDL levels 6 weeks after initiation of treatment compared to the baseline measurement (25.90±4.66 vs 12.14±2.50, P<0.05; 4.11±0.85 vsl.57±0.40, P<0.01, respectively). However, no significant effects of CCR5 overexpressing EPCs treatment on the serum level of TG and HDL were observed (1.48±1.13 vs 0.68±0.15, P>0.05; 4.18±0.38 vs 3.66±1.10, P>0.05, respectively). No significant effects of EPCs treatment (Lenti-EGFP group) on these molecules were observed. Taken together, treatment with CCR5 overexpressing EPCs ameliorates hyperlipidemia in ApoE-/- mice.5. To investigate whether the CCR5 overexpressing EPC treatment improved the endothelial cells function in ApoE-/- mice, we examined plaques content of ECs and the level of the serum Nitric Oxide (NO), a molecule with protective effects in atherosclerosis. Immunohistochemistry revealed a moderate to strong CD31+ staining in ascending arteries of Lenti-CCR5 group on week 1 and 6 post-treatment. The CD31+ staining in ascending arteries of Lenti-EGFP group was uniformly lower than in that of Lenti-CCR5 group. In addition, serum NO level was remarkably higher in the Lenti-CCR5 group than in the Lenti-EGFP group as detected by colorimetric analysis (42.3±1.8 vs.36.6±2.8, P<0.05).6. Six weeks after treatment,9 proteins were identified with significant fold changes according to the heatmap diagram. Among them, interleukin 6 (IL-6), interleukin 5 (IL-5), interleukin 3 (IL-3), interleukin 13 (IL-13), CD40 and tumor necrosis factor alpha (TNF-alpha) were decreased in the Lenti-CCR5 group. Granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein-3a (MIP-3a), and basic fibroblast growth factor (b-FGF) were slightly increased in the Lenti-CCR5 group. The IL-6 level was declined by roughly 50% in the Lenti-CCR5 group compared with that in control group. The plaques content of IL-6 was further examined by immunostaining. The positive staining area of IL-6 in the media of arteries was significantly decreased in the Lenti-CCR5 group than that in Lenti-EGFP or control group (Lenti-CCR5 vs Lenti-EGFP,13.07 ± 2.11%vs 24.3 ± 4.85%, P<0.05; Lenti-CCR5 vs control,13.07± 2.11% vs 34.03 ± 6.15%, P<0.01). However, no significant difference was found in the intima of arteries between the Lenti-CCR5 group (P=0.34) or the Lenti-EGFP (P=0.06) and the control group. The positive staining area of MMP9 was significantly decreased in the media of arteries in the Lenti-CCR5 group than that in Lenti-EGFP or control group (Lenti-CCR5 vs Lenti-EGFP,16.26±2.14% vs 23.57±0.45%, P<0.01; Lenti-CCR5 vs control,16.26±2.14% vs 30.34±2.57%, P<0.01). In the intima, there were no significant differences between the Lenti-CCR5 and Lenti-EGFP group (27.41±1.92%vs 23.42±4.58%, P=0.23). However, significantly increased MMP9 expression was found in the Lenti-CCR5 group than that in the control group (27.41±1.92% vs 10.07±3.28%, P<0.01).CONCLUSIONS1. The positive areas of CCL5 and CCR5 expression increased during progression of atherosclerotic plaques in human and mice.2. Overexpression of CCR5 increased EPC recruitment to atherosclerotic plaques.3. CCR5 overexpressing EPC treatment enhanced stability of plaques.4. CCR5 overexpressing EPC treatment decreased the serum levels of lipids.5. CCR5 overexpressing EPC treatment improved endothelial dysfunction of the atherosclerotic plaques.6. CCR5 overexpressing EPC treatment reduced pro-inflammatory factors in circulation and atherosclerotic plaques. |
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